Enzymatically hydrolyzable, serum-soluble cholesterol compounds and method for their preparation

ABSTRACT

Water and serum soluble cholesterol compounds are prepared by reactively combining certain esters of cholesterol with a solubilizing agent selected from the group consisting of peptides, proteins, water soluble polycarboxylic acids, organic and inorganic water soluble salts of said polycarboxylic acids, and cis-vicinal water-soluble polysaccharides. The water-soluble cholesterol compounds can be lyophilized and added to serum as a standard for the determination of cholesterol in biological fluids either by traditional saponofication methods or by enzymatic methods.

This is a division of application Ser. No. 716,616, filed Aug. 23, 1976,now U.S. Pat. No. 4,040,784.

FIELDS OF THE INVENTION

The fields of art to which the invention pertains include the field ofsteroid chemistry and the field of biological testing.

BACKGROUND AND SUMMARY OF THE INVENTION

In recent years, it has become common in clinical laboratories to testfor the level of cholesterol in blood serum samples. The measured levelsof cholesterol in the blood are referred to as total serum cholesterol,and include all those cholesterol compounds which are present in theblood such as cholesterol and its derivatives dihydrocholesterol and7-dihydrocholesterol, whether present in their free form or in the formof esters with the fatty acids normally present in the blood. There area number of methods which can be utilized for the determination of totalcholesterol in biological fluids. In accordance with the method of Abellet al, the serum is treated with alcoholic potassium hydroxide toliberate the cholesterol from its lipoprotein complexes and to saponifythe natural cholesterol esters. The saponified cholesterol is extractedinto a measured volume of petroleum ether and then an aliquot issubjected to color reaction utilizing a modified Libermann-Burchardreagent. Reference can be made to the journal "STANDARDIZED METHODS OFCLINICAL CHEMISTRY", vol. 2, pages 26 etc. (1958) by L. L. Abell et al.The optical density of each sample is read against a blank in aphotoelectric colorimeter. The level of cholesterol equivalent to theoptical density is calculated by comparing the optical density to thatof a standard containing a known amount of cholesterol.

For most serum clinical chemistry procedures, it is desirable to use oneserum based reference control for a variety of tests. However,cholesterol, per se, is not soluble in biological fluids such as serum.To overcome such deficiency, the prior art has attempted to solubilizethe cholesterol by forming organic salts thereof. See, for example, U.S.Pat. No. 3,859,047 to Klein. However, the level of solubility achievedis insufficient to provide sufficiently high concentrations for alldesired uses. For example, even with the use of a surfactant such asTriton X-100 (polyethyleneglycol ether of monoisoocytyl phenol, by Rohm& Haas, Inc., Philadelphia, Pa.) a useful concentration of less than 0.1gram per deciliter is provided. The sample becomes turbid atsignificantly higher concentrations, and since a colorimetric procedureis utilized, gross errors can be introduced.

More recently, a method for cholesterol analysis employing the enzymecholesterase has been developed and has been increasingly used inanalytical laboratories. Accordingly, it will be desirable for acholesterol standard to be useful not only in the saponificationchemistry of the Abell et al method but also in the enzymaticprocedures.

The present invention provides serum-soluble cholesterol compounds andmethods for their preparation, which can be lyophilized and added inhigh concentration to blood serum. A turbid-free clinical chemistrycontrol is thus provided having a high cholesterol value and which canbe assayed by the standard or enzymatic procedures. Concentration levelsof up to 1 gram of cholesterol equivalent per deciliter are easilyprepared. Furthermore, the cholesterol compounds of this invention donot interfere with other analytical tests making them highly useful in amultiple serum control.

More specifically, the present cholesterol compounds are defined bycertain esters of cholesterol reactively combined with a solubilizingagent selected from the group consisting of peptides, proteins,water-soluble polycarboxylic acids, organic and inorganic water-solublesalts of said acids, and cis-vicinal water-soluble polysaccharides. Withregard to the peptides and proteins, reaction is with the amino groupsderived therefrom (i.e., as a part thereof). The ester can be: (A) anacid ester of cholesterol, in which the acid group has at least sixcarbon atoms in its longest chain (including the acid carbon atoms),reactable with the amine component of the peptide or protein aminoresidue; or (B) an amino ester of cholesterol reactable (1) directlywith the acid portion of the peptide or protein amino residue or withthe polycarboxylic acid, (2) indirectly through a phosgene orthiophosgene coupling agent with the amine component of the peptide orprotein amino residue, or (3) indirectly through a cyanogen halide witha polysaccharide.

DETAILED DESCRIPTION

As an initial step in the preparation of a water soluble, serum-solublecholesterol compound useful herein, there is first formed an ester ofcholesterol which can be an acid ester or an amino ester, having,respectively, the following formulas: ##STR1## in which R is acholesterol radical, a is 0 or 1, n is 4-30 in formula I and is 1-30 informula II. The above esters can be prepared by reacting cholesterolwith the corresponding dibasic acid or amino acid. Examples of alkyl oraryl monoacid esters of formula (I) include cholesteryl hydrogenphthalate, cholesteryl hydrogen suberate, cholesteryl hydrogendodecanediate, and the like. Compounds such as those provided hereinwhich are formed from acid esters of cholesterol in which the acidportion contains five or less carbon atoms in its longest chain(including the acid carbon atoms) are generally not enzymaticallyhydrolyzable. The first example can be purchased from Aldrich ChemicalCo. Examples of alkyl or aryl monoamino esters of Formula (II) includecholesteryl-6'-aminohexanoate, cholesteryl-12'-aminododecanate,cholesteryl-4'-aminobenzoate, and the like.

The acid ester of cholesterol of formula (I) can be reacted directlywith the amine component of the amino residue of a protein or peptide inaccordance with the following equation. ##STR2## in which m is 1 to 100,R' is from a peptide or protein having the amino residue R'--NH₂.sbsb.m,and E is the cholesteryl ester function: ##STR3##

The amino group is from a peptide, preferably a high molecular weightpeptide, or from a protein, and in this regard it is particularlypreferred to use albumin, such as human, rabbit or bovine albumin, butpreferably the latter. Bovine albumin has 59 lysine residues, permittinga large number of cholesterol radicals to be conjugated therewith.Broadly, materials such as polylysine, polyarginine, small peptideshaving 6 or more amino acids and which contain basic groups (e.g.lysine) as a constituent, can all be used. Reference can be made to"Biological Chemistry," by H. R. Mahler and E. H. Cordes, Harper RowPub., N.Y., pages 9-120, for a description of suitable peptides, whichdescription is incorporated herein by reference.

In conducting the foregoing reaction, an activating agent should be usedand such can be defined as a material which facilitates reaction betweenan acid and an amine and which has minimum reaction with cholesterol.Such materials are well known in the field of protein and peptidesynthesis and one can refer to "BASIC PRINCIPLES OF ORGANIC CHEMISTRY"by Roberts and Cassiero, (1964), pages 702-723, published by W. A.Benjamin, Inc., incorporated herein by reference. Specific examplesinclude carbodiimides such as dicyclohexyl carbodiimide,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Woodward ReagentK-(N-ethyl-5-phenylisoxazolium-3'-sulfate), alkylchloroformate such asethylchloroformate, n-butylchloroformate, isobutylchloroformate, and thelike.

The amino ester of cholesterol of Formula (II) can be reacted directlywith the acid portion of the amino residue of a protein or peptide inaccordance with the following equation: ##STR4## in which p is 1 to 100,and R" is derived from the high molecular weight amino acid ##STR5## Asindicated, an activator is used and the reaction is conducted in a watermiscible organic solvent. The amino residue and the activator can eachbe identical to that described above with respect to the acid ester ofcholesterol of Formula (I).

The amino ester of cholesterol of Formula (II) can be reached directlywith a water-soluble polycarboxylic acid polymer, in accordance with thefollowing exemplary equation: ##STR6## in which q is 2-500,000 orhigher, q' is 2%-50% of q, R'" is hydrogen, methyl or ethyl and R^(iv)is the remaining portion of said polycarboxylic acid.

The polycarboxylic acids are preferably those which have at least one--COOH group per 20 carbon atoms of skeletal structure, and particularlyuseful materials are the polyacrylic and polymethacrylic acids. Watersoluble, inorganic salts such as those of potassium, sodium, calcium andthe like are also useful as are the water-soluble organic salts. Thelatter salts can be obtained by reaction with an organic base such asmorpholine, cyclohexylamine and tris(hydroxymethyl)aminomethane. See, inthis regard, the organic bases referred to by Klein U.S. Pat. No.3,859,047, incorporated herein by reference.

The amino ester of cholesterol of Formula (II) can be reacted by meansof a cyanogen halide coupling reagent with a cis-vicinal, water-solublepolysaccharide in accordance with the following equation: ##STR7## inwhich X is halogen such as chlorine or bromine and ##STR8## is acis-vicinal, water-soluble polysaccharide.

The amino ester of cholesterol of Formula (II) can be reacted by meansof a phosgene coupling reagent with the amine component of the aminoresidue of a protein or peptide in accordance with the followingequations: ##STR9## in which s is 1 to 100 and R^(VI) is derived fromthe high molecular weight amino acid ##STR10##

In like manner, the amino ester of cholesterol of Formula (II) can bereacted by means of a thiophosgene coupling reagent with the aminecomponent of the amino residue of a protein or peptide in accordancewith the following equation: ##STR11## in which t is 1 to 100 andR^(VII) is derived from the high molecular weight amino acid ##STR12##

In the above reactions involving phosgene or thiophosgene, the highmolecular weight amino acid can be the same as described above withrespect to reaction of the acid ester of cholesterol of Formula (I).

In each of the foregoing preparations, the peptide, protein orpolysaccharide can be dissolved in distilled water and a water-miscibleorganic solvent added thereto, such as dimethylsulfoxide, dimethylformamide, tetrahydrofuran, acetone, dioxane, acetonitrile, or the like.The cholesterol ester component and activating agent or coupling reagentcan then be added to the well mixed solution. The resultant solution canbe dialyzed against running water for several days followed bylyophilization to produce a powdery material. In general, it isdesirable to conduct the reaction between the peptide, protein orpolysaccharide and cholesterol ester component under alkalineconditions. Accordingly, a small amount of sodium hydroxide or the likecan be added to the reaction solution.

The resultant lyophilized substances can form stable aqueous solutionswhich dissolve in biological fluids such as whole serum to form clear,stable solutions yielding accurate, positive results with bothsaponification and enzymatic test procedures. The consistency of resultsis unaffected by other substances normally present in biological fluidsand the cholesterol compounds of the invention when added to biologicalfluids such as serum, do not interfere with other analytical testsusually carried out thereon thus making them highly useful as acholesterol member of multiple component analytical standards andcontrol materials. Advantageously, the cholesterol compounds of theinvention are readily soluble in water in an amount equivalent to about10 grams of cholesterol equivalent per 100 milliliter, and in biologicalfluids such as cholesterol-free serum in an amount equivalent to about 7grams of free cholesterol per 100 milliliters. For common analyticalprocedures, an amount of cholesterol compound equivalent to form about50 milligram to about 400 milligram of cholesterol per 100 milliliter,dissolved in 5 ml. water is used to reconstitute a lyophilized 5 ml.sample of the biological fluid to be tested. These amounts are chosen asthey represent the practical levels of cholesterol in the blood.Aliquots are from 0.10 to 0.50 ml. of the reconstituted serum areutilized as standards for most analytical procedures. The cholesterolcompounds of the present invention may be supplied per se in dry form oras standard stock solutions.

In the foregoing formulations, conjugated cholesterol compounds andcholesterol esters have been described wherein a is 0 or 1 and n is 4-30for acid esters and is 1-30 for amino esters. It is preferred to usesuch compounds and esters in which a is 1, n is derived from a loweralkyl group, i.e., n is 1-5 and, when a is 0, n is derived from a higheralkyl group, i.e., n is 4-30. Most preferably, a is 0 and n is derivedfrom a fatty acid group of 12-20 carbon atoms. Esters wherein a is 1 orwherein a is 0 and n is 4-30 are believed to be new compositions ofmatter.

The following examples further illustrate the invention.

EXAMPLE 1

25.8 Grams of dodecandeicarboxylic acid was dissolved in 350 ml. tolueneand placed in a three-necked flask equipped with an addition funnel,mechanical stirrer and a Dean Stark water separator. 0.1 Grams oftoluene sulfonic acid (catalytic amount) was added to the well-stirredsolution. 38.7 Grams of cholesterol, dissolved in 150 ml. toluene, wasintroduced dropwise to the refluxing solution. After the addition wascomplete, the mixture was continually heated for an additionaltwenty-four hours. Solvent was removed under vacuum. The dried materialwas dissolved in a minimum amount of tetrahydrofuran and separated asthe major component on a silica gel chromatography column to yield thedesired product.

EXAMPLE 2

20.1 Grams of 12-aminododecanoic acid can be reacted with 20.4 grams ofcarbobenzyl chloride to yield the N-protected amino acid. 15 Grams ofthe protected compound can then be dissolved in 250 ml. tetrahydrofuranand reacted with dicyclohexylcarbodiimide. The activated acid can thenbe reacted with cholesterol in equal molar concentrations to form thedesired ester. The protection group can then be removed byhydrogenolysis.

EXAMPLES 3-5

The procedure of Example 2 can be followed but substituting respectivelymolar equivalent amounts of 6-aminocaproic acid for the12-aminododecanoic acid, p-(B-aminoethyl)benzoic acid orp-(70aminoheptanyl)-benzoic acid for the 12-aminododecanoic acid.

EXAMPLE 6

3.6 grams of bovine serum albumin was dissolved in 94 ml. deionizedwater. 90 parts of dimethylformamide (DMF) was then introduced alongwith 4 ml. of 6 N sodium hydroxide.

1.6 grams of cholesteryl hydrogen dodecanedicarboxylate were dissolvedin DMF. The resulting solution was cooled by insertion into an ice bath.Then 0.556 ml. tributylamine was added, followed by 0.4 ml. ofisobutylchloroformate. This solution was stirred for 20 minutes at 4° C.The activated material was then added to the albumin solution. Theresulting solution was continuously mixed for 6 hours at ambienttemperature. A product was obtained by dialyzing for 48 hours againstdistilled water followed by lyophilization.

EXAMPLE 7

The procedure of Example 6 can be followed but substituting a molarequivalent amount of cholesteryl hydrogen phthalate for the cholesterylhydrogen dodecanedicarboxylate.

EXAMPLE 8

20 Grams of polyacrylic acid, in the form of a 25 weight percent aqueoussolution, can be mixed with a water soluble carbodiimide, in a 1/4 molarratio. After 30 minutes cholesteryl-12-aminododecanoate, in molar ratioto the carbodiimide, dissolved in dimethylformamide can be introduced.This material can then be stirred for 6 hours, dialyzed against runningdistilled water for 48 hours and lyophilized to yield the desiredproduct.

EXAMPLES 9 AND 10

The procedure of Example 8 can be followed but substituting respectivelymolar equivalent amounts of cholesteryl p-(B-aminoethyl)benzoate orp-7-aminoheptanyl benzoate.

EXAMPLE 11

3.6 Grams of bovine serum albumine (BSA) can be dissolved in 94 mls.deionized water to which solution is added 90 mls. DMF followedimmediately by 4 mls. 6 N NaOH. In another vessel with 10 ml. DMF, 3.0millimolar thiophosgene can be reacted with 1.6 grams aminocholesterolby stirring for 30-60 minutes. This solution can then be added to theBSA solution and allowed to react for four hours while maintaining analkaline pH. of 9.0 to 11.0 by appropriate addition of 6 N NaOH. Themixed solution can then be dialyzed for 48-72 hours against runningwater, and the resulting dialyzate frozen and lyophilized, to yield thedesired product.

EXAMPLE 12

1.9 grams of aminocholesterol derivative can be solubilized in 20 mls.of 75% DMF and adjusted to a pH 11.0 by addition of 6 N NaOH.

3.0 millimolar amylodextrin can be dissolved in another reagent vessel.The pH can then be rapidly adjusted to 11.0 with 6 N NaOH and thematerial allowed to sit 3 hours at ambient temperature. In anappropriate hood, 318 mg. of CNBr in 10 mls. 50% DMF can be added andallowed to react for 30 minutes while the pH is maintained at 11.0.

The cholesteryl solution can then be added to the resultant CNBramylodextrin solution and allowed to react while maintaning the pH at11.0 until the pH stabilizes. This solution can then be dialyzed againstrunning water in a hood for 48-72 hours, then frozen and lyophilized toyield the desired product.

EXAMPLE 13

In a non-enzymatic method of test, 1.5 Grams of the cholesteroldodecanedicarboxalate were added to an assayed control serum with acholesterol value of 110 msg%. Values before and after addition of thecholesterol reagent described in examples 108, 429 by the Kilaney Zak(Standard Methods in Clinical Chemistry, Ed. Meites, S. Academic PressN.Y. 1965, vol. 5, p. 79) procedure are as follows:

    ______________________________________                                        Before Addition                                                                            After Addition Recovery                                          ______________________________________                                        108          429            98                                                ______________________________________                                    

The cholesterol additive in the above cases gave a % C.V. of 2.8, asused in the method described.

EXAMPLE 14

The lyophilized cholesterol conjugate was assayed by the enzymaticprocedure outlined in the Boehringer-Mannheim product (cholesterol testcat. no. 015732).

1.5 Grams of synthesized cholesterol conjugate were added to normalassayed human serum controls and magnetically stirred to dissolve theconjugate. In each case of addition of the cholesterol conjugate, theexpected 330 mgs% increase in the cholesterol value was within + or -8%.For example, in the case where the cholesterol conjugate is described inexample no. 6, 0.375 Grams of cholesterol dodecandedicarboxylate wereadded to 25.0 ml. of 110 mg% assayed control serum. The calculatedincrease and the observed increase by the enzymatic method of B-M aredescribed in the following table:

    ______________________________________                                                    Calculated Observed                                               Determination No.                                                                         Increase   Increase  % Recovery                                   ______________________________________                                        1           330        328        99                                          2           330        335       102                                          3           330        340       103                                          ______________________________________                                    

I claim:
 1. As a new composition of matter, a serum soluble cholesterolester conjugatively coupled to the amino residue of a peptide orprotein, and having as a functional component of said ester ##STR13## inwhich R is a cholesteryl radical, a is 0 or 1, and n is 1-30 when saidester is an amino ester and is 4-30 when said ester is an acid ester,said ester being soluble in serum in an amount equivalent to form atleast 400 milligrams of cholesterol per 100 milliliters ofcholesterol-free serum.
 2. The composition of claim 1 in which saidcholesterol compound has the formula ##STR14## in which m is 1 to 100and ##STR15## is from a peptide or protein having the amino residue##STR16##
 3. The composition of claim 1 in which said cholesterolcompound has the formula ##STR17## in which p is 1 to 100 and ##STR18##is a peptide or protein having the amino residue ##STR19##
 4. Thecomposition of claim 1 in which said cholesterol compound has theformula ##STR20## in which s is 1 to 100 and ##STR21## is a peptide orprotein having the amino residue ##STR22##
 5. The composition of claim 1in which said cholesterol compound has the formula ##STR23## in which tis 1 to 100 and ##STR24## is a peptide or protein having the aminoresidue ##STR25##
 6. The composition of claim 1 wherein a is 0 and n is4-30.